Calmodulin and calmodulin kinase II mediate emergent bursting activity in the brainstem respiratory network (preBötzinger complex)

Abstract

Key points• Rhythmic network activity can be controlled by intracellular signalling pathways but their possible role is largely not yet elucidated.• This study is focused on the mechanisms of emergence and maintenance of synchronous rhythmic activity within the preBötzinger complex (preBötC), an essential part of the respiratory network in the brainstem.• Depression of the bursting activity by brief hypoxia and electrical stimulation correlated well with ADP-mediated inhibition of TRPM4 channels.• Long-lasting increases in calcium during the treatments stimulated calmodulin kinase that facilitated glutamatergic synapses and augmented rhythmic activity.• The results will help us better understand how respiratory rhythm is generated and how it is restored in various respiratory disorders Abstract Emergence of persistent activity in networks can be controlled by intracellular signalling pathways but the mechanisms involved and their role are not yet fully explored. Using calcium imaging and patch-clamp we examined the rhythmic activity in the preBötzinger complex (preBötC) in the lower brainstem that generates the respiratory motor output. In functionally intact acute slices brief hypoxia, electrical stimulation and activation of AMPA receptors transiently depressed bursting activity which then recovered with augmentation. The effects were abrogated after chelation of intracellular calcium, blockade of L-type calcium channels and inhibition of calmodulin (CaM) and CaM kinase (CaMKII). Rhythmic calcium transients and synaptic drive currents in preBötC neurons in the organotypic slices showed similar CaM-and CaMKII-dependent responses. The stimuli increased the amplitude of spontaneous and miniature excitatory synaptic currents indicating postsynaptic changes at glutamatergic synapses. In the acute and organotypic slices, CaM stimulated and ADP inhibited calcium-dependent TRPM4 channels and CaMKII augmented synaptic drive currents. Experimental data and simulations show the role of ADP and CaMKII in the control of bursting activity and its relation to intracellular signalling. I propose that CaMKII-mediated facilitation of glutamatergic transmission strengthens emergent synchronous activity within preBötC that is then maintained by periodic surges of calcium during the bursts. This may find implications in restoration and consolidation of autonomous activity in the respiratory disorders.

Expression of phospho‐Ca2+/calmodulin‐dependent protein kinase II in the pre‐Bötzinger complex of rats

“…The present morphological evidence cannot answer how NK1R/P-CaM-KII-expressed neurons in the pre-B€ otC are actually contributed to respiratory activity. However, a recent study in in vitro brainstem slice provides functional evidence that CaMKII-mediated facilitation of glutamatergic synaptic transmission strengthens synchronous bursting activity within pre-B€ otC network (Mironov 2013), which corroborates our findings.…”

CAMK2-Dependent Signaling in Neurons Is Essential for Survival

“…CAMK2 is critical for NMDA receptor-dependent signaling, and it is known that absence of the NR1 subunit of the NMDA receptor results in neonatal death due to respiratory failure in these mice (Forrest et al, 1994). CAMK2 activity mediates the emergence and maintenance of synchronous activity in the pre-Bötzinger complex, a center in the brainstem important for respiratory motor output (Mironov, 2013). Thus respiratory distress could underlie the lethality seen in the Camk2a Ϫ/Ϫ ; Camk2b Ϫ/Ϫ mutants.…”

Acute intermittent optogenetic stimulation of nucleus tractus solitarius neurons induces sympathetic long-term facilitation

“…The dorsal surface of the brain stem was exposed, and adeno-associated virus (AAV2) containing CaMKIIa-hChR2 (H134R)-mCherry (titer, 10 12 virus molecules per milliliter; purchased from the University of North Carolina vector core) was injected with a glass micropipette (25-30-m tip diameter) into the caudal NTS. CaMKIIa is expressed in glutamatergic, and not GABAergic, neurons in the thalamus and cerebral cortex (21), and CaMKII mediates emergent respiratory rhythmic discharges in glutamatergic neurons of the medulla (27). NTS injection of 200 nl of the AAV-CaMKIIa-mCherry rhodopsin vector over a 5-min period was made using the calamus scriptorius as a landmark.…”